The Experts below are selected from a list of 23922 Experts worldwide ranked by ideXlab platform

Ana Maria Cuervo - One of the best experts on this subject based on the ideXlab platform.

  • glioblastoma ablates pericytes antitumor immune function through aberrant up regulation of chaperone mediated Autophagy
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Rut Valdor, Ana Maria Cuervo, David Garciabernal, Dolores Riquelme, Carlos Martinez, Jose M Moraleda, Fernando Macian, Salvador Martinez
    Abstract:

    The contractile perivascular cells, pericytes (PC), are hijacked by glioblastoma (GB) to facilitate tumor progression. PC's protumorigenic function requires direct interaction with tumor cells and contributes to the establishment of immunotolerance to tumor growth. Cancer cells up-regulate their own Chaperone-Mediated Autophagy (CMA), a process that delivers selective cytosolic proteins to lysosomes for degradation, with pro-oncogenic effects. However, the possible impact that cancer cells may have on CMA of surrounding host cells has not been explored. We analyzed the contribution of CMA to the GB-induced changes in PC biology. We have found that CMA is markedly up-regulated in PC in response to the oxidative burst that follows PC-GB cell interaction. Genetic manipulations to block the GB-induced up-regulation of CMA in PC allows them to maintain their proinflammatory function and to support the induction of effective antitumor T cell responses required for GB clearance. GB-induced up-regulation of CMA activity in PC is essential for their effective interaction with GB cells that help tumor growth. We show that CMA inhibition in PC promotes GB cell death and the release of high immunogenic levels of granulocyte-macrophage colony stimulating factor (GM-CSF), through deregulation of the expression of cell-to-cell interaction proteins and protein secretion. A GB mouse model grafted in vivo with CMA-defective PC shows reduced GB proliferation and effective immune response compared to mice grafted with control PC. Our findings identify abnormal up-regulation of CMA as a mechanism by which GB cells elicit the immunosuppressive function of PC and stabilize GB-PC interactions necessary for tumor cell survival.

  • Chaperone-Mediated Autophagy Upregulation Rescues Megalin Expression and Localization in Cystinotic Proximal Tubule Cells
    Frontiers Media S.A., 2019
    Co-Authors: Jinzhong Zhang, Evripidis Gavathiotis, Ana Maria Cuervo, Jennifer L. Johnson, Farhana Rahman, Sergio D Catz
    Abstract:

    Cystinosis is a lysosomal storage disorder caused by defects in CTNS, the gene that encodes the lysosomal cystine transporter cystinosin. Patients with nephropathic cystinosis are characterized by endocrine defects, defective proximal tubule cell (PTC) function, the development of Fanconi syndrome and, eventually, end-stage renal disease. Kidney disease is developed despite the use of cysteamine, a drug that decreases lysosomal cystine overload but fails to correct overload-independent defects. Chaperone-Mediated Autophagy (CMA), a selective form of Autophagy, is defective in cystinotic mouse fibroblasts, and treatment with cysteamine is unable to correct CMA defects in vivo, but whether the vesicular trafficking mechanisms that lead to defective CMA in cystinosis are manifested in human PTCs is not currently known and whether PTC-specific mechanisms are corrected upon CMA upregulation remains to be elucidated. Here, using CRISPR-Cas9 technology, we develop a new human PTC line with defective cystinosin expression (CTNS-KO PTCs). We show that the expression and localization of the CMA receptor, LAMP2A, is defective in CTNS-KO PTCs. The expression of the lipidated form of LC3B, a marker for another form of Autophagy (macroAutophagy), is decreased in CTNS-KO PTCs indicating decreased autophagosome numbers under basal conditions. However, the autophagic flux is functional, as measured by induction by starvation or by blockage using the v-ATPase inhibitor bafilomycin A, and by degradation of the macroAutophagy substrate SQSTM1 under starvation and proteasome-inhibited conditions. Previous studies showed that LAMP2A accumulates in Rab11-positive vesicles in cystinotic cells. Here, we show defective Rab11 expression, localization and trafficking in CTNS-KO PTCs as determined by confocal microscopy, immunoblotting and TIRFM. We also show that both Rab11 expression and trafficking in cystinotic PTCs are rescued by the upregulation of CMA using small-molecule CMA activators. Cystinotic PTCs are characterized by PTC de-differentiation accompanied by loss of the endocytic receptor megalin, and megalin recycling is regulated by Rab11. Here we show that megalin plasma membrane localization is defective in CTNS-KO PTCs and its expression is rescued by treatment with CMA activators. Altogether, our data support that CMA upregulation has the potential to improve PTC function in cystinosis

  • transcription factor nfe2l2 nrf2 modulates chaperone mediated Autophagy through the regulation of lamp2a
    Autophagy, 2018
    Co-Authors: Marta Pajares, Antonio Diazcarretero, Ana I Rojo, Ana Maria Cuervo, Esperanza Arias, Antonio Cuadrado
    Abstract:

    Chaperone-Mediated Autophagy (CMA) is a selective degradative process for cytosolic proteins that contributes to the maintenance of proteostasis. The signaling mechanisms that control CMA are not f...

  • the coming of age of chaperone mediated Autophagy
    Nature Reviews Molecular Cell Biology, 2018
    Co-Authors: Susmita Kaushik, Ana Maria Cuervo
    Abstract:

    Chaperone-Mediated Autophagy (CMA) was the first studied process that indicated that degradation of intracellular components by the lysosome can be selective — a concept that is now well accepted for other forms of Autophagy. Lysosomes can degrade cellular cytosol in a nonspecific manner but can also discriminate what to target for degradation with the involvement of a degradation tag, a chaperone and a sophisticated mechanism to make the selected proteins cross the lysosomal membrane through a dedicated translocation complex. Recent studies modulating CMA activity in vivo using transgenic mouse models have demonstrated that selectivity confers on CMA the ability to participate in the regulation of multiple cellular functions. Timely degradation of specific cellular proteins by CMA modulates, for example, glucose and lipid metabolism, DNA repair, cellular reprograming and the cellular response to stress. These findings expand the physiological relevance of CMA beyond its originally identified role in protein quality control and reveal that CMA failure with age may aggravate diseases, such as ageing-associated neurodegeneration and cancer.

  • transcription factor nrf2 modulates chaperone mediated Autophagy through the regulation of lamp2a
    Free Radical Biology and Medicine, 2018
    Co-Authors: Marta Pajares, Antonio Diazcarretero, Ana I Rojo, Ana Maria Cuervo, Esperanza Arias, Antonio Cuadrado
    Abstract:

    Chaperone mediated Autophagy (CMA) is a selective degradative pathway in which soluble proteins are translocated into lysosomes and degraded. The regulatory mechanisms that control CMA remain largely unknown but may involve response to stress conditions. Indeed, transcriptional up-regulation of LAMP2A, the limiting step for CMA, has been reported upon oxidative stress. Considering the role of CMA in redoxtasis and proteostasis, we sought to determine if the transcription factor NRF2 has an impact on CMA modulation. We first identified and validated two NRF2 binding sequences in the LAMP2 gene and demonstrated in several human and mouse cell types that NRF2 deficiency and overexpression were linked to reduced and increased LAMP2A levels, respectively. Accordingly, lysosomal LAMP2A levels were drastically reduced in Nrf2-knockout hepatocytes, which also displayed a marked decrease in CMA activity. Oxidant challenge with paraquat or hydrogen peroxide and pharmacological activation of NRF2 with sulforaphane or dimethyl fumarate increased LAMP2A levels and CMA activity. Overall, our study identifies for the first time basal and inducible regulation of LAMP2A, and consequently CMA, by the transcription factor NRF2, pointing to a new potential strategy to combat proteinopathies.

Esperanza Arias - One of the best experts on this subject based on the ideXlab platform.

  • transcription factor nfe2l2 nrf2 modulates chaperone mediated Autophagy through the regulation of lamp2a
    Autophagy, 2018
    Co-Authors: Marta Pajares, Antonio Diazcarretero, Ana I Rojo, Ana Maria Cuervo, Esperanza Arias, Antonio Cuadrado
    Abstract:

    Chaperone-Mediated Autophagy (CMA) is a selective degradative process for cytosolic proteins that contributes to the maintenance of proteostasis. The signaling mechanisms that control CMA are not f...

  • transcription factor nrf2 modulates chaperone mediated Autophagy through the regulation of lamp2a
    Free Radical Biology and Medicine, 2018
    Co-Authors: Marta Pajares, Antonio Diazcarretero, Ana I Rojo, Ana Maria Cuervo, Esperanza Arias, Antonio Cuadrado
    Abstract:

    Chaperone mediated Autophagy (CMA) is a selective degradative pathway in which soluble proteins are translocated into lysosomes and degraded. The regulatory mechanisms that control CMA remain largely unknown but may involve response to stress conditions. Indeed, transcriptional up-regulation of LAMP2A, the limiting step for CMA, has been reported upon oxidative stress. Considering the role of CMA in redoxtasis and proteostasis, we sought to determine if the transcription factor NRF2 has an impact on CMA modulation. We first identified and validated two NRF2 binding sequences in the LAMP2 gene and demonstrated in several human and mouse cell types that NRF2 deficiency and overexpression were linked to reduced and increased LAMP2A levels, respectively. Accordingly, lysosomal LAMP2A levels were drastically reduced in Nrf2-knockout hepatocytes, which also displayed a marked decrease in CMA activity. Oxidant challenge with paraquat or hydrogen peroxide and pharmacological activation of NRF2 with sulforaphane or dimethyl fumarate increased LAMP2A levels and CMA activity. Overall, our study identifies for the first time basal and inducible regulation of LAMP2A, and consequently CMA, by the transcription factor NRF2, pointing to a new potential strategy to combat proteinopathies.

  • Transcription factor NFE2L2/NRF2 modulates Chaperone-Mediated Autophagy through the regulation of LAMP2A
    2018
    Co-Authors: Marta Pajares, Ana I Rojo, Ana Maria Cuervo, Esperanza Arias, Antonio Díaz-carretero, Antonio Cuadrado
    Abstract:

    Chaperone-Mediated Autophagy (CMA) is a selective degradative process for cytosolic proteins that contributes to the maintenance of proteostasis. The signaling mechanisms that control CMA are not fully understood but might involve response to stress conditions including oxidative stress. Considering the role of CMA in redoxtasis and proteostasis, we sought to determine if the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2) has an impact on CMA modulation. In this work, we identified and validated 2 NFE2L2 binding sequences in the LAMP2 gene and demonstrated in several human and mouse cell types that NFE2L2 deficiency and overexpression was linked to reduced and increased LAMP2A levels, respectively. Accordingly, lysosomal LAMP2A levels were drastically reduced in nfe2l2-knockout hepatocytes, which also displayed a marked decrease in CMA activity. Oxidant challenge with paraquat or hydrogen peroxide, or pharmacological activation of NFE2L2 with sulforaphane or dimethyl fumarate also increased LAMP2A levels and CMA activity. Overall, our study identifies for the first time basal and inducible regulation of LAMP2A, and consequently CMA activity, by NFE2L2. Abbreviations: ACTB: actin, beta, ARE: antioxidant response element; ATG5: Autophagy related 5; BACH1: BTB domain and CNC homolog 1; ChIP: chromatin immunoprecipitation; CMA: Chaperone-Mediated Autophagy; DHE: dihydroethidium; DMF: dimethyl fumarate; ENCODE: Encyclopedia of DNA elements at the University of California, Santa Cruz; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA: glucosylceramidase beta; GFP: green fluorescent protein; HMOX1: heme oxygenase 1; H2O2: hydrogen peroxide; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KEAP1: kelch like ECH associated protein 1; LAMP2A: lysosomal associated membrane protein 2A; LAMP2B: lysosomal associated membrane protein 2B; LAMP2C: lysosomal associated membrane protein 2C; LAMP1: lysosomal associated membrane protein 1; MAFF: MAF bZIP transcription factor F; MAFK: MAF bZIP transcription factor K; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; NQO1: NAD(P)H quinone dehydrogenase 1; PQ: paraquat; PI: protease inhibitors; qRT-PCR: quantitative real-time polymerase chain reaction; RNASE: ribonuclease A family member; SFN: sulforaphane; SQSTM1/p62: sequestosome 1; TBP: TATA-box binding protein

  • structural and biological interaction of hsc 70 protein with phosphatidylserine in endosomal microAutophagy
    Journal of Biological Chemistry, 2016
    Co-Authors: Kateryna S Morozova, Esperanza Arias, Cristina C Clement, Susmita Kaushik, Barbara Stiller, Atta Ahmad, Jennifer N Rauch, Victor Chatterjee, Chiara Melis, Brian Scharf
    Abstract:

    Abstract hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroAutophagy, Chaperone-Mediated Autophagy, and endosomal microAutophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 μm. This interaction is specific and involves a total of 4–5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microAutophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.

  • interplay of lrrk2 with chaperone mediated Autophagy
    Nature Neuroscience, 2013
    Co-Authors: Samantha J Orenstein, Esperanza Arias, Inmaculada Tasset, Hiroshi Koga, Irene Fernandezcarasa, Etty Cortes, Lawrence S Honig, William T Dauer
    Abstract:

    This study shows that Parkinson's disease–associated mutant forms of leucine-rich repeat kinase 2 (LRRK2) impair Chaperone-Mediated Autophagy in neurons, thereby reducing degradation of α-synuclein by this pathway and contributing to the accumulation of this protein observed in brain tissue from patients with Parkinson's disease.

Antonio Cuadrado - One of the best experts on this subject based on the ideXlab platform.

  • transcription factor nfe2l2 nrf2 modulates chaperone mediated Autophagy through the regulation of lamp2a
    Autophagy, 2018
    Co-Authors: Marta Pajares, Antonio Diazcarretero, Ana I Rojo, Ana Maria Cuervo, Esperanza Arias, Antonio Cuadrado
    Abstract:

    Chaperone-Mediated Autophagy (CMA) is a selective degradative process for cytosolic proteins that contributes to the maintenance of proteostasis. The signaling mechanisms that control CMA are not f...

  • transcription factor nrf2 modulates chaperone mediated Autophagy through the regulation of lamp2a
    Free Radical Biology and Medicine, 2018
    Co-Authors: Marta Pajares, Antonio Diazcarretero, Ana I Rojo, Ana Maria Cuervo, Esperanza Arias, Antonio Cuadrado
    Abstract:

    Chaperone mediated Autophagy (CMA) is a selective degradative pathway in which soluble proteins are translocated into lysosomes and degraded. The regulatory mechanisms that control CMA remain largely unknown but may involve response to stress conditions. Indeed, transcriptional up-regulation of LAMP2A, the limiting step for CMA, has been reported upon oxidative stress. Considering the role of CMA in redoxtasis and proteostasis, we sought to determine if the transcription factor NRF2 has an impact on CMA modulation. We first identified and validated two NRF2 binding sequences in the LAMP2 gene and demonstrated in several human and mouse cell types that NRF2 deficiency and overexpression were linked to reduced and increased LAMP2A levels, respectively. Accordingly, lysosomal LAMP2A levels were drastically reduced in Nrf2-knockout hepatocytes, which also displayed a marked decrease in CMA activity. Oxidant challenge with paraquat or hydrogen peroxide and pharmacological activation of NRF2 with sulforaphane or dimethyl fumarate increased LAMP2A levels and CMA activity. Overall, our study identifies for the first time basal and inducible regulation of LAMP2A, and consequently CMA, by the transcription factor NRF2, pointing to a new potential strategy to combat proteinopathies.

  • Transcription factor NFE2L2/NRF2 modulates Chaperone-Mediated Autophagy through the regulation of LAMP2A
    2018
    Co-Authors: Marta Pajares, Ana I Rojo, Ana Maria Cuervo, Esperanza Arias, Antonio Díaz-carretero, Antonio Cuadrado
    Abstract:

    Chaperone-Mediated Autophagy (CMA) is a selective degradative process for cytosolic proteins that contributes to the maintenance of proteostasis. The signaling mechanisms that control CMA are not fully understood but might involve response to stress conditions including oxidative stress. Considering the role of CMA in redoxtasis and proteostasis, we sought to determine if the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2) has an impact on CMA modulation. In this work, we identified and validated 2 NFE2L2 binding sequences in the LAMP2 gene and demonstrated in several human and mouse cell types that NFE2L2 deficiency and overexpression was linked to reduced and increased LAMP2A levels, respectively. Accordingly, lysosomal LAMP2A levels were drastically reduced in nfe2l2-knockout hepatocytes, which also displayed a marked decrease in CMA activity. Oxidant challenge with paraquat or hydrogen peroxide, or pharmacological activation of NFE2L2 with sulforaphane or dimethyl fumarate also increased LAMP2A levels and CMA activity. Overall, our study identifies for the first time basal and inducible regulation of LAMP2A, and consequently CMA activity, by NFE2L2. Abbreviations: ACTB: actin, beta, ARE: antioxidant response element; ATG5: Autophagy related 5; BACH1: BTB domain and CNC homolog 1; ChIP: chromatin immunoprecipitation; CMA: Chaperone-Mediated Autophagy; DHE: dihydroethidium; DMF: dimethyl fumarate; ENCODE: Encyclopedia of DNA elements at the University of California, Santa Cruz; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA: glucosylceramidase beta; GFP: green fluorescent protein; HMOX1: heme oxygenase 1; H2O2: hydrogen peroxide; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KEAP1: kelch like ECH associated protein 1; LAMP2A: lysosomal associated membrane protein 2A; LAMP2B: lysosomal associated membrane protein 2B; LAMP2C: lysosomal associated membrane protein 2C; LAMP1: lysosomal associated membrane protein 1; MAFF: MAF bZIP transcription factor F; MAFK: MAF bZIP transcription factor K; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; NQO1: NAD(P)H quinone dehydrogenase 1; PQ: paraquat; PI: protease inhibitors; qRT-PCR: quantitative real-time polymerase chain reaction; RNASE: ribonuclease A family member; SFN: sulforaphane; SQSTM1/p62: sequestosome 1; TBP: TATA-box binding protein

Qian Yang - One of the best experts on this subject based on the ideXlab platform.

  • essential control of mitochondrial morphology and function by chaperone mediated Autophagy through degradation of park7
    Autophagy, 2016
    Co-Authors: Bao Wang, Zhibiao Cai, Kai Tao, Weijun Zeng, Ruixin Yang, Dayun Feng, Guodong Gao, Qian Yang
    Abstract:

    As a selective degradation system, Chaperone-Mediated Autophagy (CMA) is essential for maintaining cellular homeostasis and survival under stress conditions. Increasing evidence points to an important role for the dysfunction of CMA in the pathogenesis of Parkinson disease (PD). However, the mechanisms by which CMA regulates neuronal survival under stress and its role in neurodegenerative diseases are not fully understood. PARK7/DJ-1 is an autosomal recessive familial PD gene. PARK7 plays a critical role in antioxidative response and its dysfunction leads to mitochondrial defects. In the current study, we showed that CMA mediated the lysosome-dependent degradation of PARK7. Importantly, CMA preferentially removed the oxidatively damaged nonfunctional PARK7 protein. Furthermore, CMA protected cells from mitochondrial toxin MPP(+)-induced changes in mitochondrial morphology and function, and increased cell viability. These protective effects were lost under PARK7-deficiency conditions. Conversely, overexpression of PARK7 significantly attenuated the mitochondrial dysfunction and cell death exacerbated by blocking CMA under oxidative stress. Thus, our findings reveal a mechanism by which CMA protects mitochondrial function by degrading nonfunctional PARK7 and maintaining its homeostasis, and dysregulation of this pathway may contribute to the neuronal stress and death in PD pathogenesis.

  • oxidation of survival factor mef2d in neuronal death and parkinson s disease
    Antioxidants & Redox Signaling, 2014
    Co-Authors: Wenming Li, Jin Zeng, Dean P. Jones, Qian Yang
    Abstract:

    Abstract Aims: Dysfunction of myocyte enhancer factor 2D (MEF2D), a key survival protein and transcription factor, underlies the pathogenic loss of dopaminergic (DA) neurons in Parkinson's disease (PD). Both genetic factors and neurotoxins associated with PD impair MEF2D function in vitro and in animal models of PD. We investigated whether distinct stress conditions target MEF2D via converging mechanisms. Results: We showed that exposure of a DA neuronal cell line to 6-hyroxydopamine (6-OHDA), which causes PD in animals models, led to direct oxidative modifications of MEF2D. Oxidized MEF2D bound to heat-shock cognate protein 70 kDa, the key regulator for Chaperone-Mediated Autophagy (CMA), at a higher affinity. Oxidative stress also increased the level of lysosomal-associated membrane protein 2A (LAMP2A), the rate-limiting receptor for CMA substrate flux, and stimulated CMA activity. These changes resulted in accelerated degradation of MEF2D. Importantly, 6-OHDA induced MEF2D oxidation and increased LAMP2...

  • dysregulation of Autophagy and parkinson s disease the mef2d link
    Apoptosis, 2010
    Co-Authors: Qian Yang
    Abstract:

    Autophagy refers to the process by which lysosomes degrade intracellular components. Three basic forms of it, macro-, micro-, and chaperon-mediated Autophagy, exist in cells. Several studies have shown that dysregulation of macroAutophagy compromises the viability of neurons. Recent evidence indicates that Chaperone-Mediated Autophagy plays a role in direct degradation of neuronal transcription factor MEF2D, a protein known to promote neuronal survival. Disruption of this regulatory pathway by α-synuclein leads to neuronal stress, which may underlie neuronal loss in Parkinson’s disease.

  • the complexity in regulation of mef2d by chaperone mediated Autophagy
    Autophagy, 2009
    Co-Authors: Qian Yang
    Abstract:

    Chaperone mediated Autophagy (CMA) targets specific cytoplasmic proteins for degradation by lysosomes and has been implicated to play a role in neurodegeneration.  Our recent studies identify neuronal survival factor MEF2D as a direct substrate of CMA and show that dysregulation of this process may contribute to the pathogenesis of Parkinson disease.  One interesting finding presented in our study is the apparent loss of DNA binding capacity by the accumulated MEF2D following inhibition of CMA. The possibilities and implication of this finding are discussed.

  • regulation of neuronal survival factor mef2d by chaperone mediated Autophagy
    Science, 2009
    Co-Authors: Qian Yang, Emanuela Colla, Marla Gearing, John J Shacka
    Abstract:

    Chaperone-Mediated Autophagy controls the degradation of selective cytosolic proteins and may protect neurons against degeneration. In a neuronal cell line, we found that Chaperone-Mediated Autophagy regulated the activity of myocyte enhancer factor 2D (MEF2D), a transcription factor required for neuronal survival. MEF2D was observed to continuously shuttle to the cytoplasm, interact with the chaperone Hsc70, and undergo degradation. Inhibition of Chaperone-Mediated Autophagy caused accumulation of inactive MEF2D in the cytoplasm. MEF2D levels were increased in the brains of α-synuclein transgenic mice and patients with Parkinson9s disease. Wild-type α-synuclein and a Parkinson9s disease–associated mutant disrupted the MEF2D-Hsc70 binding and led to neuronal death. Thus, Chaperone-Mediated Autophagy modulates the neuronal survival machinery, and dysregulation of this pathway is associated with Parkinson9s disease.

Evripidis Gavathiotis - One of the best experts on this subject based on the ideXlab platform.

  • Chaperone-Mediated Autophagy Upregulation Rescues Megalin Expression and Localization in Cystinotic Proximal Tubule Cells
    Frontiers Media S.A., 2019
    Co-Authors: Jinzhong Zhang, Evripidis Gavathiotis, Ana Maria Cuervo, Jennifer L. Johnson, Farhana Rahman, Sergio D Catz
    Abstract:

    Cystinosis is a lysosomal storage disorder caused by defects in CTNS, the gene that encodes the lysosomal cystine transporter cystinosin. Patients with nephropathic cystinosis are characterized by endocrine defects, defective proximal tubule cell (PTC) function, the development of Fanconi syndrome and, eventually, end-stage renal disease. Kidney disease is developed despite the use of cysteamine, a drug that decreases lysosomal cystine overload but fails to correct overload-independent defects. Chaperone-Mediated Autophagy (CMA), a selective form of Autophagy, is defective in cystinotic mouse fibroblasts, and treatment with cysteamine is unable to correct CMA defects in vivo, but whether the vesicular trafficking mechanisms that lead to defective CMA in cystinosis are manifested in human PTCs is not currently known and whether PTC-specific mechanisms are corrected upon CMA upregulation remains to be elucidated. Here, using CRISPR-Cas9 technology, we develop a new human PTC line with defective cystinosin expression (CTNS-KO PTCs). We show that the expression and localization of the CMA receptor, LAMP2A, is defective in CTNS-KO PTCs. The expression of the lipidated form of LC3B, a marker for another form of Autophagy (macroAutophagy), is decreased in CTNS-KO PTCs indicating decreased autophagosome numbers under basal conditions. However, the autophagic flux is functional, as measured by induction by starvation or by blockage using the v-ATPase inhibitor bafilomycin A, and by degradation of the macroAutophagy substrate SQSTM1 under starvation and proteasome-inhibited conditions. Previous studies showed that LAMP2A accumulates in Rab11-positive vesicles in cystinotic cells. Here, we show defective Rab11 expression, localization and trafficking in CTNS-KO PTCs as determined by confocal microscopy, immunoblotting and TIRFM. We also show that both Rab11 expression and trafficking in cystinotic PTCs are rescued by the upregulation of CMA using small-molecule CMA activators. Cystinotic PTCs are characterized by PTC de-differentiation accompanied by loss of the endocytic receptor megalin, and megalin recycling is regulated by Rab11. Here we show that megalin plasma membrane localization is defective in CTNS-KO PTCs and its expression is rescued by treatment with CMA activators. Altogether, our data support that CMA upregulation has the potential to improve PTC function in cystinosis

  • cystinosin the small gtpase rab11 and the rab7 effector rilp regulate intracellular trafficking of the chaperone mediated Autophagy receptor lamp2a
    Journal of Biological Chemistry, 2017
    Co-Authors: Jinzhong Zhang, Mahalakshmi Ramadass, Evripidis Gavathiotis, Celine J Rocca, Gennaro Napolitano, Jennifer L. Johnson, William B Kiosses, Cecilia Bucci, Jing He, Ana Maria Cuervo
    Abstract:

    Author(s): Zhang, Jinzhong; Johnson, Jennifer L; He, Jing; Napolitano, Gennaro; Ramadass, Mahalakshmi; Rocca, Celine; Kiosses, William B; Bucci, Cecilia; Xin, Qisheng; Gavathiotis, Evripidis; Cuervo, Ana Maria; Cherqui, Stephanie; Catz, Sergio D | Abstract: The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in Chaperone-Mediated Autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns-/- cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.

  • correction corrigendum chemical modulation of chaperone mediated Autophagy by retinoic acid derivatives
    Nature Chemical Biology, 2013
    Co-Authors: Jaime Anguiano, Evripidis Gavathiotis, Thomas P. Garner, Murugesan Mahalingam, Bhaskar C Das, Ana Maria Cuervo
    Abstract:

    Nat. Chem. Biol. 9, 374–382 (2013); published online 14 April 2013; corrected after print 28 June 2013 In the version of this article initially published, one of the three gray bars in Figure 6a was not defined, and the asterisks for these bars were misaligned. The errors have been corrected in the HTML and PDF versions of the article.

  • Corrigendum: Chemical modulation of Chaperone-Mediated Autophagy by retinoic acid derivatives
    Nature Chemical Biology, 2013
    Co-Authors: Jaime Anguiano, Evripidis Gavathiotis, Thomas P. Garner, Murugesan Mahalingam, Ana Maria Cuervo
    Abstract:

    Corrigendum: Chemical modulation of Chaperone-Mediated Autophagy by retinoic acid derivatives

  • Chemical modulation of Chaperone-Mediated Autophagy by retinoic acid derivatives
    Nature Chemical Biology, 2013
    Co-Authors: Jaime Anguiano, Evripidis Gavathiotis, Thomas P. Garner, Murugesan Mahalingam, Ana Maria Cuervo
    Abstract:

    Structure-based design of RARα antagonists leads to compounds that can selectively upregulate Chaperone-Mediated Autophagy (CMA), yielding the first chemically tractable target for regulating CMA in cells.